Neurotransmitter modulation of a membrane current(s) and the implications on nerve excitability has been extensively studied. This project is concerned with the modulation of the M-current, which plays a major role in the sub-threshold range for action potential initiation, causing membrane hyperpolarization and therefore, reducing cell excitability. Neurotransmitters, eg: acetylcholine and LHRH, can suppress the M-current. This produces membrane depolarization and increased input resistance, making the cell more excitable and likely to fire action potentials. Enhancing the M-current hyperpolarizes the cell and increases the stabilizing effect on membrane excitability. It is clear therefore, that modulation of the M-current by these factors places it in a unique position to control cell excitability. It is known that the M-current can be inhibited by activators of protein kinase C and enhanced by small increases of intracellular calcium. This application will use electrophysiological techniques to resolve the mechanisms involved in the modulation of the M-current. This includes how the properties of the single channels change upon current modulation and elucidation of which "messengers" may be involved in these effects. To achieve this the application's specific aims consist of two techniques. First, the technique of non-stationary ensemble variance analysis will be used to characterize the M-current, in terms of the single channel number, conductance and opening probability. Using this technique, resolution of how the M-current is suppressed by acetylcholine or LHRH will be possible, determining which channel property is changed by these agents and whether they act by the same mechanism. Once achieved, comparison with known "messengers" will be undertaken. Once this work is completed, resolution of single channel currents will be attempted using the patch-clamp technique. Patch recordings will be carried out in two configurations, "cell-attached" and excised "inside- out" modes. "Cell-attached" experiments will allow study of the modulation of M-channel activity by the agonists. If this is observed, then a soluble second messenger is implicated in this effect. If no effect is observed, excision of channel activity into the "inside-out" configuration will allow direct application o second messengers to the internal surface of the plasma membrane. If successful, a greater understanding of the role of modulation of the M-current in controlling cell activity will be attained. This information will also resolve the source of signals which can regulate the amplitude of the M-current.